Biosynthesis of Oligosaccharides

ABSTRACT

The present invention relates to methods for production of 2′Fucosyllactose by a microbial system comprising a α-1,2 fucosyltransferase (FucT2) polynucleotide and a Guanosine 5′-diphospho-β-L-fucose (GDP-L-fucose) synthesis pathway using lactose as a substrate. Furthermore, the present invention relates to compositions comprising the microbial system.

PRIORITY

This application is a continuation application of U.S. Ser. No.14/136,466, filed Dec. 20, 2013, which claims the benefit of U.S. Ser.No. 61/740,118, filed on Dec. 20, 2012, which are both incorporatedherein in their entirety by reference.

BACKGROUND

Human milk oligosaccharides (HMOs) are known to be the most relevantfactor for the development of intestinal microbiota in breast-fedinfants [1]. Also, HMOs have been reported to play important roles inpreventing adhesion of pathogens and toxins to epithelial surfaces [2].Fucosyloligosaccharides, such as 2′-fucosyllactose, lacto-N-fucopentaoseand lacto-N-difucohexaose, are common HMOs. Fucosylated oligosaccharidesact as growth stimulating factors for select Bifidobacteria and solubleanalogs of receptors for pathogenic bacteria, thereby protecting infantsagainst infection from enteric pathogens and binding of toxins [3, 4].Specifically, α-1,2-linked fucosylated oligosaccharides are reported toexhibit protective activity against several pathogens includingCampylobacter jejuni [3, 5], Salmonella enterica serotype Typhimurium[6], Enterotoxigenic E. coli [7], Helicobacter pylori [8] andnoroviruses [9]. Among them, 2′-fucosyllactose (2-FL) is typically themost abundant fucosyloligosaccharide in human milk and accounts for morethan 30% of total HMOs [3, 5]. Low levels of 2-FL in the milk of somemothers have been reported to be associated with a higher rate ofdiarrhea in breast-fed infants [3]. Hence, 2-FL is a promisingoligosaccharide for nutraceutical and pharmaceutical purposes.

SUMMARY OF THE INVENTION

The invention provides methods useful for producing 2′-fucosyllactose(2-FL) using a microbial system.

In one aspect the invention provides methods for producing2′-fucosyllactose (2-FL) in a microorganism comprising i) providing amicroorganism wherein the microorganism comprises a α-1,2fucosyltransferase (FucT2) polynucleotide and a Guanosine5′-diphospho-β-L-fucose (GDP-L-fucose) synthesis pathway; ii) fermentingthe microorganism in the presence of lactose; and iii) collecting 2-FLfrom the microorganism or from a culture broth of the microorganism.

In particular embodiments the method further comprises purifying the2-FL collected from the microorganism or from the culture broth of themicroorganism by filtering through a purification column such as anactivated charcoal and celite column.

In other particular embodiments the GDP-L-fucose synthesis pathway ismodulated for enhanced GDP-L-fucose production. For example, theGDP-L-fucose synthesis pathway is modulated by at least one ofamplification of GDP-D-mannose biosynthesis, regeneration of NADPH andmanipulation of the guanosine nucleotides biosynthetic pathway.

In other particular embodiments, the microorganism is Escherichia colior Saccharomyces cerevisiae. Suitable strains of Escherichia coliinclude the DH5α strain or JM strain. In other embodiments themicroorganism has weak ß-galactosidase activity. In yet other particularembodiments the JM strain Escherichia coli overexpresses at least one ofa phosphomannomutase (Man B) polynucleotide, a mannose 1-phosphateguanylytransferase (Man C) polynucleotide, aGDP-D-mannose-4,6-dehydratse (Gmd) polynucleotide and aGDP-4-keto-6-deoxymannose 3,5-epimerase 4-reductase (WcaG)polynucleotide.

In other particular embodiments the α-1,2 fucosyltransferasepolynucleotide is a Helicobacter pylori, Caenorhabditis elegans, Rattusnorvegicus, Mus musculus, or Homo sapien polynucleotide.

In other embodiments the presence of lactose is at a concentration ofbetween 0.5 g/l to 15 g/l. In yet other embodiments the lactose isconverted into the 2′-fucosyllactose (2-FL) at a rate of greater thanabout 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.85 or moregrams of 2-FL per gram of lactose.

In another aspect the invention provides a composition comprising arecombinant microorganism wherein the microorganism comprises amodulated GDP-L-fucose biosynthetic pathway and a α-1,2fucosyltransferase polynucleotide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an embodiment of the metabolic pathway for GDP-fucoseand 2′-fucosyllactose (2-FL) biosynthesis in recombinant E. coli. Thenames of enzymes are abbreviated as follows; ManA, mannose 6-phosphateisomerase; ManB, phosphomannomutase; ManC, mannose 1-phosphateguanylyltransferase; Gmd, GDP-D-mannose-4,6-dehydratase; WcaG,GDP-4-keto-6-deoxymannose 3,5-epimerase 4-reductase; FucT2,α-1,2-fucosyltransferase. Pi, GDP and GTP denote phosphate, guanosine5′-diphosphate and guanosine 5′-triphosphate.

FIG. 2 illustrates an embodiment of SDS-PAGE analysis of the cell crudeextract of recombinant E. coli BL21star(DE3) strains harboringpCOLADuet-1 and pHfucT2, respectively. Cells were harvested after 3 h of0.1 mM IPTG induction. T, S and I denote total, soluble and insolubleprotein fractions, respectively. The arrow indicates the correspondingprotein band with the estimated molecular weight of FucT2. Lane Mindicates size marker.

FIG. 3 illustrates an embodiment of a profile of 2-FL production in thebatch fermentation of recombinant E. coli JM109 (DE3) strain harboringplasmids pmBCGW and pHfucT2. After 3 h of 0.1 mM IPTG induction, 14.5g/l of lactose was added for 2-FL production. Symbols denote as follows;triangle, dry cell mass; diamond, 2-FL concentration; square, lactoseconcentration; circle, acetate concentration. Measurement of cell,lactose, acetate and 2-FL concentrations were done by three independentexperiments. Symbols in the figure show the representative values of thebatch fermentations.

FIG. 4 illustrates an embodiment of LC/MS analysis of 2-FL biosynthesisin the batch fermentation of the recombinant E. coli JM109 (DE3)overexpressing ManB, ManC, Gmd, WcaG and FucT2. At the end of batchfermentation, culture broth was collected for confirmation ofextracellular 2-FL production. HPLC analysis of 100 mg/l 2-FL standardsolution (A), HPLC analysis of culture broth of E. coli JM109 (DE3)harboring pmBCGW+pHfucT2 (B) and MS analysis of the compound with theretention time=6.6 min in the culture broth of E. coli JM109 (DE3)harboring pmBCGW+pHfucT2 (C).

FIG. 5 illustrates an embodiment of a calculation of the theoreticalmaximum yield of 2-FL from lactose. Elementary flux mode (EFM) analysiswas carried out for 2-FL producing E. coli.

FIG. 6 shows the plasmids and primers used in the Examples.

FIG. 7 shows the results of the batch fermentations of Example 6.

FIG. 8 illustrates an embodiment of a profile of large scale productionof 2-FL in the batch fermentation of recombinant E. coli JM109 harboringplasmids pmBCGW and pHfucT2 using a 1 L and 5 L bioreactor. Symbolsdenote as follows; square, lactose; triangle, A600 nm; diamond, 2-FLconcentration; circle, acetate concentration.

FIG. 9 illustrates the metabolic pathway for GDP-L-fucose biosynthesis.The names of enzymes are abbreviated as follows; ManA, mannose6-phosphate isomerase; ManB, phosphomannomutase; ManC, mannose1-phosphate guanylyltransferase; Gmd, GDP-D-mannose-4,6-dehydratase;WcaG, GDP-4-keto-6-deoxymannose 3,5-epimerase 4-reductase; Pi, GDP andGTP denote guanosine 5′-triphosphate. NADPH and NADP⁺ denotenicotinamide adenine dinucleotide phosphate-oxidase and nicotinamideadenine dinucleotide phosphate.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a new biosynthesis method for production of 2-FLoligosaccharides using a microbial system. In particular a method forproducing 2′-fucosyllactose (2-FL) in a microorganism, e.g., a bacteriumor yeast, comprising i) providing a microorganism wherein themicroorganism comprises a α-1,2 fucosyltransferase (FucT2)polynucleotide (e.g., a recombinant or heterologous polynucleotide) anda Guanosine 5′-diphospho-β-L-fucose (GDP-L-fucose) synthesis pathway ii)culturing the microorganism in the presence of lactose; and iii)collecting 2-FL from the microorganism or from a culture broth of themicroorganism is provided.

2-FL can be synthesized through the enzymatic fucosylation of lactose byα-1,2 fucosyltransferase (FucT2), which requires guanosine5′-diphosphate (GDP)-L-fucose as a donor of L-fucose [10]. Escherichiacoli is known to be able to synthesize GDP-L-fucose since GDP-L-fucoseis used for biosynthesis of colanic acid, one of the main components ofthe cell wall [11]. Therefore, 2-FL can be produced via engineering ofthe endogenous GDP-L-fucose biosynthetic pathway and expression oroverexpression of the fucosyltransferase polynucleotide in metabolicallyengineered bacteria such as E. coli or yeast. FIG. 1 shows the metabolicpathway for biosynthesis of GDP-L-fucose and 2-FL in recombinant E.coli. Additionally, yeasts such as Saccharomyces cerevisiae, can be usedto produce 2-FL in a similar manner as described for bacteria. S.cerevisiae makes GDP-mannose for cell wall purposes. Consequently,production of GDP-fucose in S. cerevisae requires heterologousexpression of the last two steps in the GDP-fucose biosynthesis pathwaydepicted in FIG. 1.

Enzymatic biosynthesis of fucosyloligosaccharides using a recombinantmicroorganism and fucosyltransferase has been reported. Specifically,the enzymatic synthesis of 2-FL was examined by using purified FucT2,GDP-L-fucose and lactose [10], however, the high cost of GDP-L-fucoseand FucT2 purification is a limiting factor for large-scale productionof fucosyloligosaccharides. Production of several fucose-containinglacto-oligosaccharides in recombinant E. coli was also reported throughsimultaneous overexpression of fucosyltransferase and the regulatoryprotein for colanic acid biosynthesis [12, 13]. As biosynthesis ofGDP-L-fucose, a key compound for biosynthesis of α-1,2-fucosylatedoligosaccharides, requires a number of enzymes and cofactors such asNADPH and GTP, a whole-cell conversion approach is more realistic forindustrial production than other chemical or enzymatic approaches [25].

A recombinant E. coli system for efficient production of GDP-L-fucose bymetabolic engineering was previously developed through modulation of theGDP-L-fucose synthesis pathway. An enhancement of GDP-L-fucoseproduction was achieved by modulation of several factors forbiosynthesis of GDP-L-fucose such as amplification of GDP-D-mannosebiosynthesis, regeneration of NADPH and manipulation of the guanosinenucleotides biosynthetic pathway [17-19]. Additionally, GDP-L-fucoseproduction can be maximized through optimization of fermentationconditions.

GDP-L-fucose production can be amplified, increased or modulated inbacteria and yeast by, for example, providing expression oroverexpression of one or more of ManA, ManB, ManC, Gmd, WcaGpolynucleotides by any method known in the art (e.g., providing arecombinant polynucleotide to an organism for expression of one or morepolypeptides, providing a promoter that provides for greater expressionof one or more polynucleotides, optimizing culture conditions, and anyother methods) The term “GDP-L-fucose synthesis pathway” as used hereinrefers to the metabolic pathway for GDP-L-fucose biosynthesis asillustrated in FIG. 9.

Amplification of GDP-D-mannose biosynthesis can be achieved byexpression or overexpression of the polynucleotides involved in thebiosynthesis of GDP-D-mannose such as phosphomannomutase (Man B) andmannose 1-phosphate guanylytransferase (Man C). Examples of methods forincreasing GDP-D-mannose are disclosed in [18], which is incorporated byreference herein in its entirety. Regeneration of NADPH can be achievedby overexpression of a NADPH regenerating enzyme such asglucose-6-phophate dehydrogenase, isocitrate dehydrogenase, and NADP⁺ ina recombinant microorganism. The particular methods for achievingregeneration of NADPH are disclosed in [17], which is incorporated byreference herein in its entirety. Manipulation of the guanosinenucleotides biosynthetic pathway can be achieved by increasing theintracellular levels of guanosine nucleotides and metabolic enzymesincluding inosine 5′ monophosphate dehydrogenase, guasnosine5′monophosphate synthetase, GMP reductase and guanosine-inosine kinasein a recombinant microorganism. The particular methods for achievingregeneration of NADPH are disclosed in [19], which is incorporated byreference herein in its entirety.

Each of these polynucleotides (e.g., those encoding ManA, ManB, ManC,Gmd, WcaG, glucose-6-phophate dehydrogenase, isocitrate dehydrogenase,NADP⁺, inosine 5′ monophosphate dehydrogenase, guasnosine5′monophosphate synthetase, GMP reductase and guanosine-inosine kinasecan be heterologous (e.g., from a different organism or species thanthat of the host cell) and can be present in a microbe of the inventionalone or in combination with one or more of these polynucleotides).

The term “modulation” or “modulated” as used herein refers to a change,e.g., an increase or decrease, of a cell associated activity as comparedto cell associated activity in the absence of the modulation methods.

The term “enhancement” or “enhanced” as used herein refers to increasingthe activity or concentration of GDP-L-fucose product molecules.

In the present invention, a GDP-L-fucose production system is appliedfor efficient production of 2-FL by introduction of a FucT2polynucleotide into a recombinant microorganism able to produce oroverproduce GDP-L-fucose. Suitable FucT2 polynucleotides for use in theinvention can be obtained from many organisms including, for example,from Helicobacter pylori, Caenorhabditis elegans, Rattus norvegicus, Musmusculus, or Homo sapien.

In particular embodiments the production of GDP-L-fucose is modulated bya recombinant microorganism that expresses or overexpresses enzymes thatare essential for GDP-fucose biosynthesis such asGDP-D-mannose-4,6-dehydratse (Gmd) and GDP-4-keto-6-deoxymannose3,5-epimerase 4-reductase (WcaG). Additionally, the recombinantmicroorganism can be modulated to express or overexpress enzymesinvolved in the biosynthesis of GDP-D-mannose. For example, therecombinant microorganism can be constructed for the combinationaloverexpression of enzymes such as phosphomannomutase (Man B) and mannose1-phosphate guanylytransferase (Man C). The overexpression of theenzymes can be achieved by constructing inducible overexpression vectorsencoding for the desired enzyme. The particular methods for producingrecombinant microorganisms that overexpress GMD, WcaG, Man B and Man Care disclosed in [18], which is incorporated by reference herein in itsentirety. Suitable recombinant microorganisms include but are notlimited to bacteria such as E. coli and yeast such as S. cerevisiae.

The term “overexpression” or “overexpressed” as used herein refers to alevel of enzyme or polypeptide expression that is greater than what ismeasured in a wild-type cell of the same species as the host cell thathas not been genetically altered.

The methods of the invention provide for the production of 2-FL in theamount of 0.01 g/L to 4.35 g/L. For example, 2-FL is produced using arecombinant or mutant microorganism that overexpresses FucT2 (e.g., 0.01g/L of 2-FL produced). Additionally, 2-FL is produced from a recombinantor mutant microorganism that is optimized for 2-FL production because ituses just enough lactose to achieve growth, and conserves most lactosefor use in making 2-FL (e.g., 0.15 g/L of 2-FL produced). Moreover, 2-FLis produced by culturing a microorganism expressing or overexpressingFucT2 and has a modulated or amplified GDP-L-fucose biosynthetic pathwayunder optimized batch fermentation conditions (e.g., 1.23 g/L of 2-FL)or under fed-batch fermentation conditions (e.g., 4.35 g/L of 2-FLproduced). Therefore, 2-FL can be produced at about 0.005, 0.01, 0.05,0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 or more g/L.

Optimized fermentation conditions, such as batch fermentation conditionsinclude regulation of the culture temperature and the lactoseconcentration. For example, the culture can be maintained at atemperature wherein the substrate is efficiently used and 2-FL ismaximally produced with minimal acetate production. Specifically, theculture temperature is maintained between about 25° C. and about 37° C.with the optimal temperature being about 25° C. Additionally, underoptimal conditions the lactose substrate concentration is between 0.5g/L to 50 g/L. In particular embodiments the lactose is added at aconcentration of about 0.5 g/L, 10 g/L, 20 g/L, 30 g/L, 40 g/L, 50 g/Lor more. In other embodiments 2-FL production is performed usingfed-batch fermentation wherein lactose is continuously suppliedthroughout the fermentation process at a concentration of about 5, 10,15, 20, 25, 30, 35, 40 or more g/L.

Expression or overexpression of the amplified or modulated GDP-L-fucosepathway in combination with a FucT2 polynucleotide provides a systemwherein 2-FL is synthesized within one cell. Whole cell biosynthesis of2-FL from lactose can be assessed in a series of batch fermentations forrecombinant microbes expressing or overexpressing the necessary genesfor GDP-L-fucose overproduction and FucT2. An EFM analysis for 2-FLproduction in the recombinant E. coli can also be used to compare andevaluate experimental results. See Appendix. To construct an efficient2-FL production system by metabolic engineering, an understanding anddetailed analysis of a cellular metabolic network involved in the 2-FLbiosynthesis is important. Elementary flux mode (EFM) analysis hasemerged as a powerful tool for metabolic pathway analysis. EFM analysisis a useful mathematical tool for defining and describing all metabolicroutes that are both stoichiometrically and thermodynamically feasiblefor a group of enzymes. The EFM analysis can decompose a complexmetabolic network of many highly interconnected reactions into uniquelyorganized pathways that support steady state of metabolism. EFM analysiscan provide identification of all genetically independent pathways,determination of the most efficient physiological state of a cell, andanalysis of metabolic network properties such as robustness andregulation [14-16]. Hence, it can be a useful tool for understandingdynamics of cellular metabolism and rational design of the host strain'smetabolism for 2-FL production.

In one aspect, a method of the invention includes, fermenting therecombinant microorganism overexpressing the GDP-L-fucosepolynucleotides (e.g., one or more of polynucleotides capable ofexpressing ManA, ManB, ManC, Gmd, WcaG, glucose-6-phophatedehydrogenase, isocitrate dehydrogenase, NADP⁺, inosine 5′ monophosphatedehydrogenase, guasnosine 5′monophosphate synthetase, GMP reductase andguanosine-inosine kinase) and a FucT2 polynucleotide in the presence ofa lactose substrate. The use of lactose as a substrate for the methodsdescribed herein is advantageous over previous methods using glucosebecause it allows for the production of 2-FL by a whole-cell conversionapproach rather than a chemical or enzymatic approach. The whole-cellconversion approach provides for efficient production of 2-FL directlywithout the need for any costly starting materials such as purifiedGDP-L-fucose. Instead, the whole-cell approach derives all the necessarymaterials for the production of the 2-FL directly from the recombinantmicroorganism. Due to catabolite repression, lactose transport isrepressed when glucose is used as a substrate. Thus, 2-FL productionusing glucose cannot be achieved using a whole-cell conversion approachbecause GDP-L-fucose must first be produced in a cell and thenenzymatically added to lactose.

In one embodiment of the invention the culture medium has less thanabout 20, 10, 5, 4, 3, 2, 1 or less g/L of glucose.

The microorganism used in the methods described herein can be unable toassimilate lactose or to utilize lactose extremely inefficiently due to,for example, a partial deletion or inactivation of one or more lacZgenes, which code for β-galactosidase such that the microorganismproduces 20, 30, 40, 50, 60, 70, 80, 90, 95, 99% or less β-galactosidasethan a microorganism with a non-defective lacZ gene. In particularembodiments the microorganism has weak β-galactosidase activity ascompared to a microorganism that has a non-defective lacZ gene. The term“weak β-galactosidase activity” as used herein refers to a cell that hasresidual β-galactosidase activity which provides the cell only enoughlactose to survive on.

Examples of suitable microorganisms include S. cerevisiae and E. colistrains, such as DH5α and JM series. In one embodiment the microorganismis a slow-lactose utilization microorganism wherein it uses just enoughlactose to achieve growth, and conserves most lactose for use in making2-FL. Several attempts for production of fucosylated (or sialylated)oligosaccharides from lactose have been made using the derivative of E.coli JM107 and JM109 since these strains are unable to produce an activeβ-galactosidase due to the insertion of the M15 single strand DNA intothe lacZ gene [12, 13, 26, 27]. In these cases, glucose (or glycerol)was used as another carbon source for GDP-fucose production (orCMP-N-acetylneuraminic acid). These nucleotide sugars are subsequentlyused for fucosylation (or sialylation).

Surprisingly, it was determined that microorganisms that produce lessβ-galactosidase than a microorganism with a non-defective lacZ gene arethe best producers of 2-FL (as compared to microorganisms that expressnormal levels of ß-galactosidase or express no 1-galactosidase) usingthe methods of the invention. E. coli JM109(DE3) was chosen as anexample of a suitable host strain for 2-FL production.

In one aspect, a method of the invention includes collecting 2-FL fromthe microorganism or from a culture broth of the microorganism.Following collection, the 2-FL is purified using a purification columnssuch as an activated charcoal and celite column.

Polynucleotides

Polynucleotides contain less than an entire microbial genome and can besingle- or double-stranded nucleic acids. A polynucleotide can be RNA,DNA, cDNA, genomic DNA, chemically synthesized RNA or DNA orcombinations thereof. The polynucleotides can be purified free of othercomponents, such as proteins, lipids and other polynucleotides.

Polynucleotides can be isolated from nucleic acid sequences present in,for example, a bacterial or yeast culture. Polynucleotides can also besynthesized in the laboratory, for example, using an automaticsynthesizer. An amplification method such as PCR can be used to amplifypolynucleotides from either genomic DNA or cDNA encoding thepolypeptides.

Polynucleotides of the invention can comprise coding sequences fornaturally occurring polypeptides or can encode altered sequences that donot occur in nature. If desired, polynucleotides can be cloned into anexpression vector comprising expression control elements, including forexample, origins of replication, promoters, enhancers, or otherregulatory elements that drive expression of the polynucleotides of theinvention in host cells. An expression vector can be, for example, aplasmid, such as pBR322, pUC, or ColE1, or an adenovirus vector, such asan adenovirus Type 2 vector or Type 5 vector, but any kind of vector canbe used.

Methods for preparing polynucleotides operably linked to an expressioncontrol sequence and expressing them in a host cell are well-known inthe art. See, e.g., U.S. Pat. No. 4,366,246. A polynucleotide of theinvention is operably linked when it is positioned adjacent to or closeto one or more expression control elements, which direct transcriptionand/or translation of the polynucleotide. Polynucleotides of theinvention can be present in an expression vector, which can be presentin a host cell.

Polynucleotides can encode full-length polypeptides, polypeptidefragments, and variant or fusion polypeptides. A polynucleotide encodesa polypeptide, which can be an enzyme that has biological activity.

A polypeptide expressed by a polynucleotide of the invention reactssubstantially the same as a wild-type polypeptide in an assay ofbiological activity, e.g. has 80-120% of the activity of the wild-typepolypeptide. A wild-type polypeptide is a polypeptide that is notgenetically altered and that has an average biological activity in anatural population of the organism from which it is derived.

A polypeptide of the invention can be produced recombinantly. Apolynucleotide encoding a polypeptide of the invention can be introducedinto a recombinant expression vector, which can be expressed in asuitable expression host cell system using techniques well known in theart. A variety of bacterial, yeast, plant, mammalian, and insectexpression systems are available in the art and any such expressionsystem can be used.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which a disclosed disclosure belongs.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise.

The disclosure may be further understood by the following non-limitingexamples. Although the description herein contains many specificities,these should not be construed as limiting the scope of the disclosurebut as merely providing illustrations of some of the presently preferredembodiments of the disclosure. For example, thus the scope of thedisclosure should be determined by the appended aspects and theirequivalents, rather than by the examples given.

All references throughout this application, for example patent documentsincluding issued or granted patents or equivalents; patent applicationpublications; and non-patent literature documents or other sourcematerial; are hereby incorporated by reference herein in theirentireties, as though individually incorporated by reference, to theextent each reference is at least partially not inconsistent with thedisclosure in this application (for example, a reference that ispartially inconsistent is incorporated by reference except for thepartially inconsistent portion of the reference).

The terms and expressions which have been employed herein are used asterms of description and not of limitation, and there is no intention inthe use of such terms and expressions of excluding any equivalents ofthe features shown and described or portions thereof, but it isrecognized that various modifications are possible within the scope ofthe disclosure. Thus, it should be understood that although the presentdisclosure has been specifically disclosed by preferred embodiments,exemplary embodiments and optional features, modification and variationof the concepts herein disclosed may be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this disclosure as defined by the appended aspects.The specific embodiments provided herein are examples of usefulembodiments of the present disclosure and it will be apparent to oneskilled in the art that the present disclosure may be carried out usinga large number of variations of the devices, device components, methodssteps set forth in the present description. As will be obvious to one ofskill in the art, methods and devices useful for the present methods caninclude a large number of optional composition and processing elementsand steps.

Whenever a range is given in the specification, for example, atemperature range, a time range, or a composition or concentrationrange, all intermediate ranges and subranges, as well as all individualvalues included in the ranges given are intended to be included in thedisclosure. It will be understood that any subranges or individualvalues in a range or subrange that are included in the descriptionherein can be excluded from the aspects herein.

In each instance herein any of the terms “comprising”, “consistingessentially of” and “consisting of” may be replaced with either of theother two terms. The disclosure illustratively described herein suitablymay be practiced in the absence of any element or elements, limitationor limitations which is not specifically disclosed herein.

EXAMPLES Example 1: Strains and Plasmids

E. coli TOP10 [F-mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 recA1araD139 Δ(ara-leu) 7697 galU galK rpsL (Str^(R)) endA1 nupG] was usedfor genetic manipulation. E. coli BL21star(DE3) [F⁻, ompT, hsdSB(r_(B)⁻m_(B) ⁻), gal, dcm me131 (DE3)] (Invitrogen, Carlsbad, Calif., USA) andJM109(DE3) [endA1 glnV44 thi-1 relA1 gyrA96 recA1 mcrB⁺ Δ(lac-proAB)e14-[F′ traD36 proAB⁺ lacl^(q) lacZΔM15] hsdR17(r_(K) ⁻m_(K) ⁺) (DE3)](NEB, Ipswich, Mass., USA) were used for production of GDP-L-fucose and2-FL.

Plasmid pmBCGW containing the polycistronic gmd-wcaG gene cluster andmanB-manC gene cluster was previously constructed using plasmidpETDuet-1 [18]. The gene encoding FucT2 was obtained by the polymerasechain reactions (PCR) using the genomic DNA of the Helicobacter pylori26695 strain (ATCC 700392) as template [20]. Two PCR primers of fucT2_Fand fucT2_R were used for the amplification of the FucT2 gene. Afterdigestion of PCR fragments of the FucT2 gene and pCOLADuet-1 (MerckBiosciences, Darmstadt, Germany) with NcoI and SacI, the DNA fragmentswere ligated to construct plasmid pHfucT2. Plasmids and primers used inthis work are shown in FIG. 6. The constructed plasmid was confirmed byDNA sequencing. The conditions for PCR reaction, DNA manipulation andbacterial transformation followed the descriptions in [21].

Example 2: Batch Fermentation

Batch fermentation was carried out in a 250 ml flask containing 50 ml ofLB medium at 25° C. and pH 6.8. Agitation speed was maintained at 250rpm. When dry cell mass reached 0.3 g/l, 0.1 mMisopropyl-β-D-thiogalactopyranoside (IPTG) was added to culture broth.After 3 h of additional cultivation, 2.6 g/l (or 14.5 g/l) lactose wasadded for 2-FL production.

Example 3: Analytical Methods

Cell concentration was measured by optical density (OD) at 600 nm usinga spectrophotometer (Biomate 5, Thermo, NY, USA). Overexpression ofFucT2 inside the cell was analyzed by using sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 12%polyacrylamide). After 3 h of 0.1 mM IPTG induction, cells werecollected and the concentration was adjusted to around 7.2 g/l. Theywere resuspended in 50 mM potassium phosphate buffer (pH 7.0) anddisrupted by an ultrasonic processor. After centrifugation at 15,000×gfor 20 min, the supernatant (soluble fraction) and debris (insolublefraction) were separated. Ten microliters of the soluble proteinfraction (approximately 0.04 mg) and the same volume of the total andinsoluble protein fractions were subjected to SDS-PAGE. Gels werestained with Coomassie brilliant blue solution and images were analyzedusing a densitometer.

Concentrations of lactose, 2-FL and acetate in batch fermentations weredetermined by using a high performance liquid chromatography (HPLC)system (Agilent Technologies 1200 Series) equipped with a Rezex ROAOrganic Acid H⁺ column (Phenomenex, Torrance, Calif., USA) and arefractive index (RI) detector (Agilent, Palo Alto, Calif., USA). Thecolumn was eluted with 0.01N H₂SO₄ at a flow rate of 0.6 ml/min at 50°C.

In order to confirm 2-FL biosynthesis, culture broth at the end of thebatch fermentation was collected and analyzed using a liquidchromatography/mass spectrometry (LC/MS) system. The LC (AgilentTechnologies 1100 Series) was equipped with an Agilent Zorbax EclipseZDB-C8 (4.6×150 mm, 5 micron) column and an Agilent LC/MSD Trap XCT Plusdetector. The column was eluted at a flow rate of 0.4 ml/min by thefollowing gradient program: 95% (v/v) eluent A (15 mM ammonium acetate)and 5% eluent B (acetonitrile) for 1 min; 5% to 95% eluent B over 6 min;95% eluent B over 10 min. The scan range for MS was 70-600mass-to-charge ratio (m/z).

Example 4: Construction of Metabolic Network Model for E. coli Producing2-FL from Lactose

A metabolic network model was constructed for 2-FL producing E. colithat grows on lactose. The E. coli network was based on a model that wasintroduced by Stelling et al. [22] to examine the relationship betweenstructure and function in metabolic networks. Furthermore, the model hasbeen used for calculating elementary flux modes in previous reports [23,24]. The metabolic network was composed of 108 reactions, which wereinvolved in carbon central metabolism, amino acid synthesis, fatty acidsynthesis and biomass production (Supplementary information: METATOOLinput file). The catabolic part of the model included substrate uptakereactions, glycolysis, pentose phosphate pathway, TCA cycle, andexcretion of by-products (e.g. acetate, formate, lactate, and ethanol).Previous networks were extended to include the anaplerotic reactions(e.g. malic enzyme and pyruvate oxidase) in addition to parallelpathways for initial acetate metabolism. The anabolic part of the modelcovers the conversion of precursors into building blocks likemacromolecules and biomass. The core E. coli model from Stelling et al.[22] was modified in this research to account for lactose consumptionand synthesis of 2-FL. Among the reactions added for 2-FL synthesis,some minor adjustments were made to simplify the model. Lactose wasassumed to break down to 2 moles of glucose because galactose can beeasily converted into glucose-6-phosphate. ATP was used in place of GTPfor energy transfer. As for the mass balance, it should be noted thatADP is formed whenever ATP is consumed for all the metabolic reactionsin the network. The mass balance equation on ATP is therefore thenegative of the mass balance on ADP and thus the two equations arelinearly dependent. Therefore, ADP can be excluded from the model inorder to simplify the subsequent EFM calculation. The same is true forother cofactor pairs like NADP/NADPH and NAD/NADH. The EFM pathways inthe model were estimated using METATOOL 5.1 [14, 16] with Matlab.

Example 5: Expression of α-1,2-Fucosyltransferase (FucT2) in RecombinantE. coli

The expression pattern of FucT2 was investigated during a batchfermentation of recombinant E. coli harboring plasmid pHfucT2. The FucT2polynucleotide from H. pylori was cloned and overexpressed in the E.coli BL21star(DE3) strain. In order to maximize the expression of thesoluble form of FucT2 in the recombinant E. coli, 0.1 mM of IPTG wasused. As shown in FIG. 2, a 33 kDa protein (consistent with FucT2, [20])was found in both soluble and insoluble fraction. While a significantamount of FucT2 was expressed in inclusion bodies, biosynthesis of 2-FLwas expected because a soluble form of FucT2 was available as well.

Example 6: Batch Fermentations

From the preliminary experiments (whole cell bioconversion of 2 g/llactose with E. coli BL21star(DE3) strain), it was concluded that the E.coli BL21star(DE3) strain was not beneficial for 2-FL production becauseit consumed lactose for growth and maintenance instead of converting to2-FL (data not shown). Most of the initially added lactose was consumedwithin 12 h of fermentation with a marginal growth during thefermentation (data not shown). Some E. coli strains, such as DH5α and JMseries, are known to be unable to assimilate lactose or utilize lactoseextremely inefficiently due to partial deletion of the lacZ gene, whichcodes for β-galactosidase. As such, these E. coli strains are useful for2-FL production. Hence, E. coli JM109(DE3) enabling overexpressingproteins under the control of T7 promoter was used as an alternativehost stain for 2-FL production.

2-FL production for BL21star(DE3) and JM109(DE3) was compared underbatch fermentation conditions. In order to allow sufficient productionof both GDP-L-fucose biosynthetic enzymes and FucT2 inside the cells,the cells were cultivated for 3 hours after 0.1 mM IPTG induction. Then,2.6 g/l of lactose was added to initiate 2-FL production withoutaddition of additional sugar because GDP-L-fucose can be produced fromLB media [17, 18]. During the fermentations, extracellular 2-FLproduction (in the medium) was monitored by HPLC analysis. As a result,a small amount of 2-FL (10 mg/l) was produced in the batch fermentationof recombinant E. coli BL21star(DE3). Meanwhile, much higher amount of2-FL was produced in the batch fermentation of recombinant E. coliJM109(DE3). About 140 mg/l of 2-FL was produced from 2.6 g/l of lactosewhile 0.4 g/l of lactose remained unused at the end of the fermentation(data not shown). These results indicate that the lactose concentrationshould be controlled at more than 0.5 g/l to maintain 2-FL production.Consequently, a yield of 60 mg 2-FL/g lactose was obtained from thebatch fermentation of E. coli JM109(DE3) when 2.6 g/l of lactose wasused. In order to obtain a higher amount of 2-FL, a batch fermentationwith a higher concentration of lactose was carried out. FIG. 3 shows theprofiles of lactose consumption and 2-FL production in the batchfermentation of recombinant E. coli JM109(DE3) with 14.5 g/l lactose.The cells consumed lactose slowly but produced 2-FL constantly for 96hours. After 96 h of fermentation, the 2-FL concentration did notincrease any further and lactose consumption stopped. As a result, amaximum 2-FL concentration of 1.23 g/l was obtained, which correspondedto a nine-fold (1.23 g/l vs. 140 mg/l) increase as compared with theprevious fermentation with 2.6 g/l lactose. 2-FL yield increased to 90mg 2-FL/g lactose when 14.5 g/l of lactose was used. The results of thebatch fermentations are shown in FIG. 7.

It is generally known that acetate formation is accelerated when themetabolic fluxes to pyruvate exceed the capacity of the respiratorymetabolism [28, 29]. Slow consumption of lactose might not lead toacetate formation, suggesting that the lactose utilization rate by E.coli JM109(DE3) is not fast enough to cause acetate formation.

Example 7: Confirmation of 2-FL Biosynthesis by Recombinant E. coliOverexpressing GDP-L-Fucose Biosynthetic Enzymes and FucT2

LC/MS analysis was performed to confirm the biosynthesis of 2-FL in therecombinant E. coli JM109(DE3) strain overexpressing ManB, ManC, Gmd,WcaG and FucT2. HPLC data showed that a compound with the identicalretention time to 2-FL was detected in the culture broth (FIG. 4B). MSscanning data (compound with RT=6.6 min) showed ion fragment of m/z487.1, which is compatible with 2-FL (FIG. 4C).

Example 8: Evaluation of 2-FL Yield Using EFM Analysis for 2-FLProducing E. coli from Lactose

In order to evaluate the efficiency of 2-FL production from lactoseusing the recombinant E. coli JM109(DE3) strain, elementary flux mode(EFM) analysis was employed to estimate a maximum theoretical yield of2-FL from lactose. FIG. 5 shows the prediction of theoretical 2-FL yieldversus biomass yield for E. coli growing on lactose. The experimentalresult from a batch fermentation of 14.5 g/l of lactose resulted in abiomass yield of 0.1 g biomass/g lactose. This suggests that 2-FLproduction from lactose by the engineered E. coli reached 20% of themaximum 2-FL production capacity.

This result indicates that more than 90% of lactose consumed was usedfor other purposes such as biomass production and endogeneousmetabolism. Slow consumption of lactose was also observed in the batchfermentation with mixed sugars (2 g/l of lactose and 5 g/l of mannose),where it was expected that lactose could be mainly used for 2-FLproduction as mannose might be used for cell growth. Although anenhancement of 2-FL yield (0.13 g 2-FL/g lactose) was obtained, most ofthe consumed lactose was not used for 2-FL production (data not shown).

Example 9: Purification of 2-FL

The 2-FL produced from lactose using the recombinant E. coli JM109(DE3)strain was collected from the fermentation supernatant by filtering thesupernatant through an activated charcoal and celite column.Specifically, the LB fermentation media was applied to the activatedcarbon celite filter (1:1) and washed with 5% ethanol. The 2-FL was theneluted with 30% ethanol and the ethanol was allowed to evaporate.

Chemical analysis using MS of the purified sample confirmed an ionfragment of 501.775 m/z, which is consistent with 2-FL.

Example 10: Large Scale Production of 2-FL

The methods of the invention were applied to a large scale production of2-FL using a bioreactor. Specifically, recombinant JM109(DE3) wasfermented in a 1 L and 5 L bioreactor at a concentration of 15 g/L oflactose at 25° C. Under these conditions, the fermentation in the 1 Land 5 L reactors produced ˜1.5 g/L. The results of the batchfermentations are shown in FIG. 8.

REFERENCES

-   1. Kunz C, Rudloff S: Health promoting aspects of milk    oligosaccharides. International Dairy Journal 2006, 16(11):    1341-1346.-   2. Bode L: Recent advances on structure, metabolism, and function of    human milk oligosaccharides. Journal of Nutrition 2006,    136(8):2127-2130.-   3. Morrow A L, Ruiz-Palacios G M, Altaye M, Jiang X, Guerrero M L,    Meinzen-Derr J K, Farkas T, Chaturvedi P, Pickering L K, Newburg D    S: Human milk oligosaccharides are associated with protection    against diarrhea in breast-fed infants. Journal of Pediatrics 2004,    145(3):297-303.-   4. Newburg D S, Ruiz-Palacios G M, Altaye M, Chaturvedi P,    Meinzen-Derr J, Guerrero M D, Morrow A L: Innate protection    conferred by fucosylated oligosaccharides of human milk against    diarrhea in breastfed infants. Glycobiology 2004, 14(3):253-263.-   5. Chaturvedi P, Warren C D, Altaye M, Morrow A L, Ruiz-Palacios G,    Pickering L K, Newburg D S: Fucosylated human milk oligosaccharides    vary between individuals and over the course of lactation.    Glycobiology 2001, 11(5):365-372.-   6. Chessa D, Winter M G, Jakomin M, Bäumler A J: Salmonella enterica    serotype Typhimurium Std fimbriae bind terminal α(1,2)fucose    residues in the cecal mucosa. Molecular Microbiology 2009,    71(4):864-875.-   7. Newburg D S, Pickering L K, McCluer R H, Cleary T G: Fucosylated    oligosaccharides of human milk protect suckling mice from    heat-stabile enterotoxin of Escherichia coli. Journal of Infectious    Diseases 1990, 162(5): 1075-1080.-   8. Maqalhães A, Reis C A: Helicobacter pylori adhesion to gastric    epithelial cells is mediated by glycan receptors. Brazilian Journal    of Medical and Biological Research 2010, 43(7):611-618.-   9. Newburg D S, Ruiz-Palacios G M, Morrow A L: Human milk glycans    protect infants against enteric pathogens. Annual Review of    Nutrition 2005, 25:37-58.-   10. Albermann C, Piepersberg W, Wehmeier U F: Synthesis of the milk    oligosaccharide 2′-fucosyllactose using recombinant bacterial    enzymes. Carbohydrate Research 2001, 334(2):97-103.-   11. Stevenson G, Andrianopoulos K, Hobbs M, Reeves P R: Organization    of the Escherichia coli K-12 gene cluster responsible for production    of the extracellular polysaccharide colanic acid. Journal of    Bacteriology 1996, 178(16):4885-4893.-   12. Drouillard S, Driguez H, Samain E: Large scale synthesis of H    antigen oligosaccharides by expressing Helicobacter pylori al,    2-fucosyltransferase in metabolically engineered Escherichia coli    cells. Angewandte Chemie 2006, 118(11):1810-1812.-   13. Dumon C, Priem B, Martin S L, Heyraud A, Bosso C, Samain E: In    vivo fucosylation of lacto-N-neotetraose and lacto-N-neohexaose by    heterologous expression of Helicobacter pylori α-1, 3    fucosyltransferase in engineered Escherichia coli. Glycoconjugate    Journal 2001, 18(6):465-474.-   14. Pfeiffer T, Sánchez-Valdenebro I, Nuño J C, Montero F, Schuster    S: METATOOL: for studying metabolic networks. Bioinfomatics 1999,    15(3):251-257.-   15. Schuster S, Fell D A, Dandekar T: A general definition of    metabolic pathways useful for systematic organization and analysis    of complex metabolic networks. Nature Biotechnology 2000,    18(3):326-332.-   16. Trinh C T, Wlaschin A, Srienc F: Elementary mode analysis: a    useful metabolic pathway analysis tool for characterizing cellular    metabolism. Applied Microbiology and Biotechnology 2009,    81(5):813-826.-   17. Lee W H, Chin Y W, Han N S, Kim M D, Seo J H. Enhanced    production of GDP-L-fucose by overexpression of NADPH regenerator in    recombinant Escherichia coli. Applied Microbiology and Biotechnology    2011, 91(4):967-976.-   18. Lee W H, Han N S, Park Y C, Seo J H: Modulation of guanosine    5′-diphosphate-D-mannose metabolism in recombinant Escherichia coli    for production of guanosine 5′-diphosphate-L-fucose. Bioresource    Technology 2009, 100(24):6143-6148.-   19. Lee W H, Shin S Y, Kim M D, Han N S, Seo J H: Modulation of    guanosine nucleotides biosynthetic pathways enhanced GDP-L-fucose    production in recombinant Escherichia coli. Applied Microbiology and    Biotechnology 2012, 93(6):2327-2334.-   20. Wang G, Boulton P G, Chan N W, Palcic M M, Taylor D E: Novel    Helicobacter pylori α1,2-fucosyltransferase, a key enzyme in the    synthesis of Lewis antigens. Microbiology 1999, 145(11):3245-3253.-   21. Byun S G, Kim M D, Lee W H, Lee K J, Han N S, Seo J H:    Production of GDP-L-fucose, L-fucose donor for    fucosyloligosaccharide synthesis, in recombinant Escherichia coli.    Applied Microbiology and Biotechnology 2007, 74(4):768-775.-   22. Stelling J, Klamt S, Bettenbrock K, Schuster S, Gilles E D:    Metabolic network structure determines key aspects of functionality    and regulation. Nature 2002, 420(6912): 190-193.-   23. Klamt S, Gagneur J, von Kamp A: Algorithmic approaches for    computing elementary modes in large biochemical reaction networks.    IEE Proceedings Systems Biology 2005, 152(4):249-255.-   24. Urbanczik R, Wagner C: An improved algorithm for stoichiometric    network analysis: theory and applications. Bioinformatics 2005,    21(7):1203-1210.-   25. Ruffing A, Chen R R: Metabolic engineering of microbes for    oligosaccharide and polysaccharide synthesis. Microbial Cell    Factories 2006, 5:25.-   26. Dumon C, Samain E, Priem B: Assessment of the two Helicobacter    pylori α-1, 3 fucosyltransferase ortholog genes for the large scale    synthesis of LewisX human milk oligosaccharides by metabolically    engineered Escherichia coli. Biotechnology Progress 2004,    20(2):412-419.-   27. Fierfort N, Samain E: Genetic engineering of Escherichia coli    for the economical production of sialylated oligosaccharides.    Journal of Biotechnology 2008, 134(3-4):261-265.-   28. Lee S Y: High cell-density culture of Escherichia coli. Trends    in Biotechnology 1996, 14(3):98-105.-   29. Zhao J, Baba T, Mori H, Shimizu K: Global metabolic response of    Escherichia coli to gnd or zwf gene-knockout, based on ¹³C-labeling    experiments and the measurement of enzyme activities. Applied    Microbiology and Biotechnology 2004, 64(1):91-98.

APPENDIX METATOOL Input File ENZREV

CO2_ex G6P::F6P F16P::T3P DHAP::G3P G3P::DPG DPG::3PG 3PG::2PG 2PG::PEPCit::ICit ICit::alKG SuccCoA::Succ Fum::Mal Mal::OxA G6P::PGlacAcCoA::Adh Adh::Eth R15P::X5P R15P::R5P Transket1 Transaldo Transket2AcCoA::AcP AcP::Ac Pyr::Lac NADHDehydro TransHydro ATPSynth MTHF_Synth

ENZIRREV

mue O2_up N_up S_up Glc_PTS Glc_ATP DHAP::Glyc3P Lac_ex Lact_up LactaseFL_rxn Eth_ex Ac_ex Form_ex F16P::F6P F6P::F16P PEP::PYR Pyr::PEPPYR::AcCoA AcCoA::Cit alKG::SuccCoA Succ::Fum Fum::Succ ICit::GlyoxGlyox::Mal PGlac::PGluc PGluc::R15P OxA::PEP PEP::OxA Pyr::Form OxidaseATPdrain Chor_Synth PRPP_Synth Ala_Synth Val_Synth Leu_Synth Asn_Synth_1Asp_synth Asp::Fum Asp::AspSAld AspSAld::HSer Lys_Synth Met_SynthThr_Synth Ile_Synth His_Synth Glu_synth Gln_Synth Pro_Synth Arg_SynthTrp_Synth Tyr_Synth Phe_Synth Ser_Synth Gly_Synth Cys_Synth rATP_SynthrGTP_Synth rCTP_Synth rUTP_Synth dATP_Synth dGTP_Synth dCTP_SynthdTTP_Synth mit_FS_Synth UDPGlc_Synth CDPEth_Synth OH_myr_ac_SynthC14_0_FS_Synth CMP_KDO_Synth NDPHep_Synth TDPGlcs_Synth UDP_NAG_SynthUDP_NAM_Synth di_am_pim_Synth ADPGlc_Synth Mal::Pyr Pyr::Ac Ac::AcCoA

METINT

Gluc Lact G6P F6P F16P DHAP Glyc3P G3P DPG 3PG 2PG PEP Pyr AcCoA CitICit alKG SuccCoA Succ Fum Mal OxA Glyox R5P R15P E4P X5P S7P PGlacPGluc ATP NADH NADPH QH2 Hp O2 CO2 N S AcP Ac Form Lac Adh Eth Chor PRPPMTHF AspSAld HSer Ala Cys Asp Glu Phe Gly His Ile Lys Leu MetAsn Pro Gln Arg Ser Thr Val Trp Tyr rATP rGTP rCTP rUTP dATP dGTP dCTPdTTP mit_FS UDPGlcCDPEth OH_myr_ac C14_0_FS CMP_KDO NDPHep TDPGlcs UDP_NAG UDP_NAMdi_am_pim ADPGlc

METEXT

O2_ext N_ext CO2_ext Lac_ext Lact_ext Eth_ext Ac_ext Form_ext ATP_extFL_ext Biomass

CAT

mue: 0.14176 Glyc3P+26.2949 ATP+0.60097 Ala+0.10124 Cys+0.26647Asp+0.30747 Glu+0.2048 Phe+0.67725 Gly+0.10473 His+0.32116 Ile+0.37935Lys+0.49804 Leu+0.16989 Met+0.26647 Asn+0.24436 Pro+0.29091 Gln+0.32698Arg+0.38031 Ser+0.28044 Thr+0.46778 Val+0.062835 Trp+0.15244 Tyr+0.1489rATP+0.18319 rGTP+0.11366 rCTP+0.12273 rUTP+0.023904 dATP+0.024582dGTP+0.024582 dCTP+0.023904 dTTP+0.28352 mit_FS+0.0069264UDPGlc+0.010368 CDPEth+0.010368 OH_myrac+0.010368 C14_0_FS+0.010368CMP_KDO+0.010368 NDPHep+0.0069264 TDPGlcs+0.01656 UDP_NAG+0.01656UDP_NAM+0.01656 di_am_pim+0.0924 ADPGlc=Biomass.O2_up: 1 O2_ext=1 O2.

N_up: 1 Next=1 N. CO2 ex: 1 CO2=1 CO2 ext. S_up: 4 ATP+4 NADPH=1 S.Glc_PTS: 1 Gluc+1 PEP=1 G6P+1 Pyr. Glc_ATP: 1 Gluc+1 ATP=1 G6P.DHAP::Glyc3P: 1 DHAP+1 NADH=1 Glyc3P.

Lac_ex: 1 Lac=1 Lac_ext.Lact_up: 1 Lact_ext=1 Lact.

Lactase: 1 Lact=2 Gluc.

FL_rxn: 1 G6P+1 ATP+1 NADPH+1 Lact=1FL_ext.Eth_ex: 1 Eth=1 Eth_ext.Ac_ex: 1 Ac=1 Ac_ext.Form_ex: 1 Form=1 Form_ext.

G6P::F6P: 1 G6P=1 F6P. F16P::F6P: 1 F16P=1 F6P. F6P::F16P: 1 F6P+1 ATP=1F16P. F16P::T3P: 1 F16P=1 DHAP+1 G3P. DHAP::G3P: 1 DHAP=1 G3P. G3P::DPG:1 G3P=1 DPG+1 NADH. DPG::3PG: 1 DPG⁼1 3PG+1 ATP. 3PG::2PG: 1 3PG=1 2PG.2PG::PEP: 1 2PG=1 PEP. PEP::PYR: 1 PEP=1 Pyr+1 ATP. Pyr::PEP: 1 Pyr+2ATP=1 PEP. PYR::AcCoA: 1 Pyr=1 AcCoA+1 NADH+1 CO2. AcCoA::Cit: 1 AcCoA+1OxA=1 Cit. Cit::ICit: 1 Cit=1 ICit.

ICit::alKG: 1 ICit=1 alKG+1 NADPH+1 CO2.alKG::SuccCoA: 1 alKG=1 SuccCoA+1 NADH+1 CO2.

SuccCoA::Succ: 1 SuccCoA=1 Succ+1 ATP. Succ::Fum: 1 Succ=1 Fum+1 QH2.Fum::Succ: 1 Fum+1 QH2=1 Succ. Fum::Mal: 1 Fum=1 Mal. Mal::OxA: 1 Mal=1OxA+1 NADH. ICit::Glyox: 1 ICit=1 Succ+1 Glyox. Glyox::Mal: 1 AcCoA+1Glyox=1 Mal. G6P::PGlac: 1 G6P=1 PGlac+1 NADPH. AcCoA::Adh: 1 AcCoA+1NADH=1 Adh. Adh::Eth: 1 NADH+1 Adh=1 Eth. PGlac::PGluc: 1 PGlac=1 PGluc.PGluc::R15P: 1 PGluc=1 R15P+1 NADPH+1 CO2. R15P::X5P: 1 R15P=1 X5P.R15P::R5P: 1 R15P=1 R5P. Transket1: 1 R5P+1 X5P=1 G3P+1 S7P. Transaldo:1 G3P+1 S7P=1 F6P+1 E4P. Transket2: 1 E4P+1 X5P=1 F6P+1 G3P. OxA::PEP: 1OxA+1 ATP=1 PEP+1 CO2. PEP::OxA: 1 PEP+1 CO2=1 OxA. AcCoA::AcP: 1AcCoA=1 AcP. AcP::Ac: 1 AcP=1 ATP+1 Ac. Pyr::Form: 1 Pyr=1 AcCoA+1 Form.Pyr::Lac: 1 Pyr+1 NADH=1 Lac. NADHDehydro: 1 NADH=1 QH2+2 Hp. Oxidase: 1QH2+0.5 02=2 Hp. TransHydro: 1 NADH+1 Hp=1 NADPH. ATPSynth: 3 Hp=1 ATP.ATPdrain: 1 ATP=1 ATP_ext. Chor_Synth: 2 PEP+1 E4P+1 ATP+1 NADPH=1 Chor.PRPP_Synth: 1 R5P+2 ATP=1 PRPP. MTHF_Synth: 1 ATP+1 NADPH=1 MTHF.

Ala_Synth: 1 Pyr+1 Glu=1 alKG+1 Ala.Val_Synth: 2 Pyr+1 NADPH+1 Glu=1 alKG+1 CO2+1 Val.Leu_Synth: 2 Pyr+1 AcCoA+1 NADPH+1 Glu=1 alKG+1 NADH+2 CO2+1 Leu.Asn_Synth_1: 2 ATP+1 N+1 Asp=1 Asn.Asp_synth: 1 OxA+1 Glu=1 alKG+1 Asp.

Asp::Fum: 1 Asp=1 Fum+1 N. Asp::AspSAld: 1 ATP+1 NADPH+1 Asp=1 AspSAld.AspSAld::HSer: 1 NADPH+1 AspSAld=1 HSer.

Lys_Synth: 1 di_am_pim=1 CO2+1 Lys.

Met_Synth: 1 SuccCoA+1 MTHF+1 HSer+1 Cys=1 Pyr+1 Succ+1 N+1 Met.Thr_Synth: 1 ATP+1 HSer=1 Thr.

Ile_Synth: 1 Pyr+1 NADPH+1 Glu+1 Thr=1 alKG+1 CO2+1 N+1 Ile.His_Synth: 1 ATP+1 PRPP+1 Gln=1 alKG+2 NADH+1 His.Glu_synth: 1 alKG+1 NADPH+1 N=1 Glu.

Gln_Synth: 1 ATP+1 N+1 Glu=1 Gln. Pro_Synth: 1 ATP+2 NADPH+1 Glu=1 Pro.

Arg_Synth: 1 AcCoA+4 ATP+1 NADPH+1 CO2+1 N+1 Asp+2 Glu=1 alKG+1 Fum

+1 Ac+1 Arg Trp_Synth: 1 Chor+1 PRPP+1 Gln+1 Ser=1 G3P+1 Pyr+1 CO2+1Glu+1 Trp.

Tyr_Synth: 1 Chor+1 Glu=1 alKG+1 NADH+1 CO2+1 Tyr.Phe_Synth: 1 Chor+1 Glu=1 alKG+1 CO2+1 Phe.Ser_Synth: 1 3PG+1 Glu=1 alKG+1 NADH+1 Ser.

Gly_Synth: 1 Ser=1 MTHF+1 Gly. Cys_Synth: 1 AcCoA+1 S+1 Ser=1 Ac+1 Cys.

rATP_Synth: 5 ATP+1 CO2+1 PRPP+2 MTHF+2 Asp+1 Gly+2 Gln=2 Fum+1 NADPH+2Glu+1 rATP.rGTP_Synth: 6 ATP+1 CO2+1 PRPP+2 MTHF+1 Asp+1 Gly+3 Gln=2 Fum+1 NADH+1NADPH+3 Glu+1 rGTP.rCTP_Synth: 1 ATP+1 Gln+1 rUTP=1 Glu+1 rCTP.rUTP_Synth: 4 ATP+1 N+1 PRPP+1 Asp=1 NADH+1 rUTP.dATP_Synth: 1 NADPH+1 rATP=1 dATP.dGTP_Synth: 1 NADPH+1 rGTP=1 dGTP.dCTP_Synth: 1 NADPH+1 rCTP=1 dCTP.dTTP_Synth: 2 NADPH+1 MTHF+1 rUTP=1 dTTP.mit_FS_Synth: 8.24 AcCoA+7.24 ATP+13.91 NADPH=1 mit_FS.

UDPGlc_Synth: 1 G6P+1 ATP=1 UDPGlc. CDPEth_Synth: 1 3PG+3 ATP+1 NADPH+1N=1 NADH+1 CDPEth.

OH_myr_ac_Synth: 7 AcCoA+6 ATP+11 NADPH=1 OH_myr_ac.C14_0_FS_Synth: 7 AcCoA+6 ATP+12 NADPH=1 C14_0_FS.CMP_KDO_Synth: 1 PEP+1 R5P+2 ATP=1 CMP_KDO.

NDPHep_Synth: 1.5 G6P+1 ATP=4 NADPH+1 NDPHep. TDPGlcs_Synth: 1 F6P+2ATP+1 N=1 TDPGlcs.

UDP_NAG_Synth: 1 F6P+1 AcCoA+1 ATP+1 Gln=1 Glu+1 UDP_NAG.UDP_NAM_Synth: 1 PEP+1 NADPH+1 UDP_NAG=1 UDP_NAM.di_am_pim_Synth: 1 Pyr+1 SuccCoA+1 NADPH+1 AspSAld+1 Glu=1 alKG+1 Succ+1di_am_pim.

ADPGlc_Synth: 1 G6P+1 ATP=1 ADPGlc. Mal::Pyr: 1 Mal=1 Pyr+1 NADH+1 CO2.Pyr::Ac: 1 Pyr=1 QH2+1 CO2+1 Ac. Ac::AcCoA: 2 ATP+1 Ac=1 AcCoA.

We claim:
 1. A method for producing 2′-fucosyllactose (2-FL) in amicroorganism, comprising i) providing a microorganism wherein themicroorganism comprises a α-1,2 fucosyltransferase (FucT2)polynucleotide and a Guanosine 5′-diphospho-β-L-fucose (GDP-L-fucose)synthesis pathway; ii) fermenting the microorganism in the presence oflactose; and iii) collecting 2-FL from the microorganism or from aculture broth of the microorganism.
 2. The method of claim 1 wherein theGDP-L-fucose synthesis pathway is modulated for enhanced GDP-L-fucoseproduction.
 3. The method of claim 2 wherein the GDP-L-fucose synthesispathway is modulated by at least one of amplification of GDP-D-mannosebiosynthesis, regeneration of NADPH and manipulation of the guanosinenucleotides biosynthetic pathway.
 4. The method of claim 1 wherein themicroorganism is a bacteria or yeast.
 5. The method of claim 4 whereinthe bacteria is Escherichia coli and the yeast is Saccharomycescerevisiae.
 6. The method of claim 5 wherein the Escherichia coli isDH5α strain or JM strain.
 7. The method of claim 6 wherein theEscherichia coli is JM strain.
 8. The method of claim 7 wherein the JMstrain Escherichia coli overexpresses at least one of aphosphomannomutase (Man B) polynucleotide, a mannose 1-phosphateguanylytransferase (Man C) polynucleotide, aGDP-D-mannose-4,6-dehydratse (Gmd) polynucleotide and aGDP-4-keto-6-deoxymannose 3,5-epimerase 4-reductase (WcaG)polynucleotide.
 9. The method of claim 1 wherein the α-1,2fucosyltransferase polynucleotide is a Helicobacter pylori,Caenorhabditis elegans, Rattus norvegicus, Mus musculus, or Homo sapienpolynucleotide.
 10. The method of claim 1 wherein the presence oflactose is at a concentration of between 0.5 g/l to 15 g/l.
 11. Themethod of claim 1, wherein the lactose is converted into the2′-fucosyllactose (2-FL) at a rate of greater than about 0.1 g of 2-FLper gram of lactose.
 12. The method of claim 1, wherein themicroorganism has weak ß-galatosidase activity.
 13. A compositioncomprising a recombinant microorganism wherein the microorganismcomprises a modulated GDP-L-fucose biosynthetic pathway and a α-1,2fucosyltransferase polynucleotide.
 14. The composition of claim 13wherein the GDP-L-fucose synthesis pathway is modulated by at least oneof amplification of GDP-D-mannose biosynthesis, regeneration of NADPHand manipulation of the guanosine nucleotides biosynthetic pathway. 15.The composition of claim 13 wherein the microorganism is a bacteria oryeast.
 16. The composition of claim 15 wherein the bacteria isEscherichia coli and the yeast is Saccharomyces cerevisiae.
 17. Thecomposition of claim 16 wherein the Escherichia coli is DH5α strain orJM strain.
 18. The composition of claim 17 wherein the Escherichia coliis JM strain.
 19. The composition of claim 18 wherein the JM strainEscherichia coli overexpresses at least one of a phosphomannomutase (ManB) polynucleotide, a mannose 1-phosphate guanylytransferase (Man C)polynucleotide, a GDP-D-mannose-4,6-dehydratse (Gmd) polynucleotide anda GDP-4-keto-6-deoxymannose 3,5-epimerase 4-reductase (WcaG)polynucleotide.
 20. The composition of claim 13 wherein the α-1,2fucosyltransferase polynucleotide is a Helicobacter pylori,Caenorhabditis elegans, Rattus norvegicus, Mus musculus, or Homo sapienpolynucleotide.